CD1d stimulates NKT cells that are thought to be immune regulatory T cells that regulate a number of autoimmune and tumor conditions. Unknown autologous lipids may be involved in the CD1 d-mediated stimulation of NKT cells, and this stimulation can be enhanced by a synthetic glycolipid, a-galatosyl ceramide (a-GalCer). To understand the CD1 -mediated presentation of lipids, we propose to investigate the molecular and cellular basis of this pathway, using the NKT cell recognition of a-GalCer plus mouse CD1d (mCDld). We will analyze the intracellular trafficking of antigens and mCD1 d, glycolipid antigen processing, and the in vivo effects on NKT cells resulting from the alteration of the antigen processing pathway. In the first aim, we will express mutant mCD1 d molecules with restricted intracellular trafficking, and carry out cell fractionation studies to determine where processing and mCD1 d antigen binding occur. In the second aim, we will determine the in vivo effect of altering mCD1 d intracellular trafficking on the development and function of NKT cells. The results from these studies are also expected to help in the design of lipid antigen-based vaccines and in attempts to alter immune regulation by stimulating NKT cells.